Microsynth, one of Letgen Biotechnology's key partners, is one of Europe's leading companies, especially in the field of nucleic acid synthesis and analysis.
If you are performing quantitative PCR (qPCR), it is very important to know and understand what the ct value is. In this month's newsletter we will discuss the critical role of the ct value in qPCR, its changing nomenclature, how it is calculated and what happens when things sometimes go wrong.
All Nomenclature
We would like to emphasize that a ct value has been given many names over the years as technology has developed and changed, including the ones listed below. These values are all the same, although their meaning may be a bit strange when translated into Turkish. To standardize qPCR terminology, MIQE (Minimum Information guidelines for the Publication of Quantitative Real-Time PCR Assays) recommends the use of the Cq value.
Ct - (threshold cycle) threshold cycle
Cp - (crossing point) crossing point
TOP - (take-off point) take-off point
Cq - (quantification cycle) quantification cycle
What is this Cq Value?
Real-time PCR (qPCR) is used to measure the absolute amount of a target sequence or to compare the relative amounts of a target sequence between samples. This technique is monitored in real time by amplification of a specific fluorescent signal that binds to the target sequence. Although fluorescent probes are sequence-specific, background fluorescence can also occur during most qPCR experiments. It is crucial to be able to ignore or account for these background signals if we want to gather meaningful information about the target sequence. This problem is addressed by two factors in qPCR: Threshold line and Cq value.
Threshold is the point at which the target sequence reaches a detection-level fluorescence intensity. It is the signal that reflects a statistically significant increase, which can be calculated automatically or manually.
The Cq value is the number of PCR cycles where the reaction curve of the sample intersects the threshold line. The qPCR instrument software calculates and plots the Cq value for each sample. Cq is used to calculate the starting DNA copy number because the Cq value is inversely proportional to the starting amount of the target. At the end of each cycle your qPCR instrument will collect the fluorescence data and the fluorescence signal that you see as a straight line from the first PCR cycle until about 15 PCR cycles will start to show logarithmic amplification, rising above the threshold line after the 15th cycle.
Figure. Threshold level and Cq-value in a real-time PCR amplification curve.
Cq Factors affecting the value of
There are many factors that can influence this value.
- Fluorescence emission can be affected by pH and salt concentration in a solution. You should therefore ensure that you use high-quality PCR components that are not expired and have been stored under appropriate conditions.
- If you are using passive reference dyes such as ROX , the ratio of ROX to FAM dye will determine the reaction values. A low amount of ROX produces a high reaction value.
- PCR efficiency depends on the specificity of the primers, the temperature of the primer Tm and the initial concentration and purity of the sample. An efficiency above 90% is acceptable for PCR efficiency. We can detect a perfect PCR when the cq difference between 10-fold dilutions is approximately 3.3 cycles.
- Again , a correlation coefficient (R²) of 1 indicates that the PCR study was excellent.
- When working on the standard curve, working at least 4 points with 3 repetitions will provide you with the healthiest results.
- For RNA/cDNA studies, you should keep the area sterile at all times to avoid RNAse contamination and avoid freeze-thawing samples.
References
1. Bustin SA, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009. 55(4):611-22. doi: 10.1373/clinchem.2008.112797.
2. C. Schrader, et al. PCR inhibitors - occurrence, properties and removal. J AppliedMicrobiology. 113(5):1014-26. doi: 10.1111/j.1365-2672.2012.05384.x
We have talked about what plasmids are and their applications in previous newsletters. Let's take a closer look at plasmid copy numbers and the ways they can be useful in the lab.
The contents of this month's newsletter;
- What exactly does plasmid copy number mean?
- Why is plasmid copy number important?
- It will be on how the plasmid copy number can be changed.
Plasmid copy number can have a big impact on yield, and while sometimes it is best to have a high plasmid DNA copy number, other times it can be a disadvantage.
What is Plasmid Copy Number?
Plasmid copy number refers to the number of molecules of a single plasmid present in the host bacterial cell. They are expressed as low (15-20 copies per cell), medium (20-100 copies per cell) or high (500-700 copies per cell) copy numbers and vary in size depending on three main factors.
1) Ori and its components (e.g. ColE1 RNA I and RNA II).
2) The size of the plasmid and its associated attachment (larger attachments and plasmids can be replicated in a lower number as they represent an enormous metabolic burden for the cell).
3) Culture conditions (factors affecting the metabolic load on the host).
Why Knowing the Copy Number of Your Plasmid is Important
It is very important to know which category your plasmid falls into before you start working. If you know that you are working with a low copy number plasmid, you should not be too surprised by the low yield and you can decide to create more culture media.
One advantage of a high plasmid copy number is the higher stability of the plasmid. But this is not to say that the opposite does not happen!
When is a High Plasmid Copy Number Important?
- If you are experiencing low protein yields when performing protein production studies, a high copy number of plasmids, possibly due to a high metabolic load, can lead to protein aggregation and insufficient modification.
- Using high plasmid copy numbers in cloning studies will result in higher yields.
When is Low Plasmid Copy Number Good?
- While a high copy number is always considered good, some situations are better suited to a low copy number. For example, if you want to study a fungal protein for its antibacterial properties, it might be good to work with a low plasmid copy number to minimize toxic effects and not kill the bacteria.
- Overexpressed proteins, incorrect protein-protein pairings can lead to structural problems in the protein itself.
How do we change the plasmid copy number?
For the reasons mentioned above, it can be advantageous to have different plasmids with different copy numbers to choose from when doing your studies. Detailed studies on plasmid replication have paved the way for how we should vary plasmid copy number:
- You can increase the copy number of some plasmids by growing the host bacteria at high temperatures.
- Exposure of the host bacterium to chloramphenicol inhibits bacterial protein synthesis, leading to chromosomal replication inhibition and cell division inhibition. Plasmids require long-lived proteins and continue to replicate even when replication and cell division have stopped. Eventually, when the environment runs out of proteins, plasmid replication will stop, but the average copy number will have increased significantly.
qPCRis very popular as a qualitative and quantitative detection tool. Often you need to use a standard curve to get reliable, reproducible data. qPCRLet us examine together the whys and hows of the standard curve:
qPCR looks like a very simple technique at first glance, and when optimized, it gives great results in your experiment. To ensure that we get consistent and accurate results that reflect what is really happening in the sample under study, we must use the right controls. One of these basic controls is to create a standard curve plot. The standard curve plot allows us to check the efficiency of our primers and the quality of the DNA.
Perfect qPCR Efficient Primers are Important for their Data:
You have designed a primer on the computer with the help of a bioinformatics software, you have seen with your own eyes that it is specific through the NCBI portal, you have confidently ordered the primers you have designed to be synthesized, you have received your primers, it is imperative to test them with a qPCR experiment to avoid making mistakes, getting wrong results and wasting time! This is a big risk that no researcher wants to take. Don't be impatient to do the study right away and make sure to set up an experimental design for your first study!
It is important to make sure that the ct values obtained are accurate and reflect reality. The logarithmic curves you get with a single dilution, which you think look good, are not always accurate and do not guarantee efficient replication. This is why you should always test your primers with a standard curve. A standard curve plot can show that the primers bind efficiently and precisely to the target and elongate correctly, which is a critical point.
One qPCR How is the Standard Curve Realized?
We use the quantities of at least 5 different data points of the same DNA sample diluted 10-fold to generate a standard curve plot. Theoretically, correctly designed efficient primers should result in a dose-response curve in direct proportion. You then need to plot the Ct values on the y-axis and the log DNA copies per mL on the x-axis. You can prepare a very simple Excel document for this. Below is an example of a standard curve.
The second important issue is to work in at least 3 replicates so that we can also evaluate reproducibility, it will be much more accurate to obtain precise results. One last footnote is to pipette DNA that you are sure of its purity in each reaction and do not forget to use a negative control to eliminate the contamination problem.
Analyzing Your Standard Curve
Some qPCR software has applications for analyzing the standard curve, but many instruments do not have this option. When analyzing your curve, there are a few things to consider when calculating and evaluating it:
PCR Efficiency
Considering that the DNA molecule doubles every cycle, the PR efficiency range should be between 90% and 110%. Why do we give this range of values? Because reactions are never perfect. If some mistakes are made due to dilutions, the efficiency will go above 100%. For values below 90%, we can interpret that you have inhibitor contamination or low primer yield.
R² Value
The R² value is the correlation coefficient and should > be 0.99 to ensure good confidence in the correlation.
This value allows you to see if there is a good linear relationship between the values of each sample. A low value may indicate a poor serial dilution. To avoid this, when making your serial dilutions, make sure you pipette correctly using well-calibrated pipettes and mix the dilutions thoroughly.
Cq Standard Deviation for Values
Performing the amplification helps to reduce errors and makes your R² and PCR efficiency values more reliable. However, if there is too much variability in your replicates, we cannot say that they are reliable values.
To check how reliable your copies are, calculate the standard deviation (SD). Good copies should be within 0.2 SD units. If they are not, you may need to redo your standard curve.
If you get good data from your qPCR standard curve, you are well on your way to being satisfied with the efficiency of your primers! But what if your standard curve data is not so good? Here are two options:
What if your Primers are not Efficient?
It happens surprisingly often that the qPCR standard curve plot ends up lopsided; each DNA concentration results in approximately the same Ct value. This means that the primers are not binding efficiently to the target.
A Bad One qPCR Standard Curve May Reflect Low DNA Expression
A bad standard curve may not be due to primers. If your target is poorly expressed in your sample, your standard curve may be incorrect. You need to verify whether this target is expressed in the cell type you are working with. If your target is poorly expressed, increase the amount of DNA used for amplification. Or vice versa... This can help you determine the appropriate amount of DNA to use in your next experiments. Why use 10 ng per reaction when you can get good amplification at 1 ng? This way you can use your very valuable DNA samples in your different qPCR runs.
It is often tempting to skip the qPCR standard curve step, but in the long run this step can be good for assessing primer efficiency and DNA quality.
Taking the time at the beginning to ensure your primers are efficient can save a lot of time in the long run and ultimately lead to better results!
References
1. Korenkova V, et al. (2015) Pre-amplification in the context of high-throughput qPCRagain expression experiment. BMC Mol Biol. 16:5.
2. Van Peer G, Mestdagh P, Vandesompele J. (2012) Accurate RT-qPCR again expressionanalysis on cell culture lysates. Sci Rep. 2:222.
Exosomes are small extracellular vesicles that are released from cells throughout the body and enable intercellular communication. They carry various structures specific to the cell from which they are released. These include specific microRNAs, proteins and metabolites.
Exosomes can circulate in the body with the help of various fluids. Examples of these fluids are blood, saliva, breast milk, amniotic fluid, cerebrospinal fluid and urine .
These fluids can be used in exosome research and their exact function can be revealed by exosome isolation.
Okay. exosomes how does he know what to carry to which cell?
Although the exact target cell-exosome relationship is not known, researchers believe that this relationship is ensured by special protein markers on the surface of exosomes.
There is no universal method to purify exosomes from body fluids or culture media. While ultracentrifugation was once the traditional method, several alternative methods are now available.
Exosomes can be purified by different methods:
Ultracentrifugation
: separates exosomes from other substances in the environment with a centrifuge capable of spinning samples at extremely high speed.
Polymer Precipitation: separates exosomes from the solution using polymers (such as PEG).
Size Exclusion Chromatography: samples are passed through a column containing a stationary phase with a known pore size.
Immunoaffinity
: uses antigens to bind specific proteins on the surface of the exosome
Ultrafiltration:
the sample is passed through a filter with a known molecular weight value
Silicon Carbide: Based on Norgen's patented resin method that binds exosomes under specific pH conditions
Norgen quickly and easily from a variety of sample types. exosome provides a purification method.
Under specific buffer conditions, exosomes, Silicon Carbide resin and are thus obtained with a high degree of purity and specificity.
Once exosomes have been purified, various methods of analysis are needed to understand what they contain and what function they provide. Some of these include
Analysis
Transmission Electron Microscopy (TEM)
To visualize exosome morphology
RNA Isolation and Next Generation Sequencing
To identify the RNA species present in the exosome
Mass Spectrometry
It can be used to identify and characterize proteins on and within exosomes.
Downstream Applications
Cosmetics
Exosomes are used in research in the dermatological and cosmetic industries, from topical wound healing to potential skin and hair rejuvenation effects.
Medicine Transportation
Due to their natural occurrence in the body, cell specificity and ability to cross biological barriers, exosomes show promise as a novel drug delivery system.
Disease Monitoring
By analyzing the substances carried by exosomes, biomarkers can be created to help monitor disease development, progression and treatment effect.
Good yields are vital in plasmid isolation. How do you optimize your preparation to achieve a good plasmid yield and what causes low yields? We address these questions and much more below:
What is the best yield during plasmid isolation?
There are three main types of plasmid preparation: mini, midi and maxi. The amount of plasmid DNA varies between protocols and kits, but the table below provides an average range for these three different preparation methods.
Prep Type. | Plasmid DNA Recovery (µg) |
Mini Prep | 550 |
Midi Prep | 50-200 |
Maxi Prep | 200-1000 |
**When both samples are processed at the same time and in the same way, if one sample yields well and the other plasmid yields poorly, what could be the problem? Many things can cause yield differences between plasmid preparations. Let's unravel this mystery item by item and see what we can do.
1. Problematic Inserts
You prepared two plasmids at the same time using the same protocol and got different yields. If the plasmids have the same "backbone" then the cause could be an insert. Some inserts can be problematic for bacteria, for example a protein that makes them sick, or unstable, such as repetitive sequences. You can try using specialized cell lines to overcome the problem.
2. Number of Copies
The second important point is how the insert size changes the copy number of the plasmid. Large inserts will reduce the copy number of the plasmid, meaning you will need to grow more cells to get a good yield.
If genes are cloned into different vectors, the problem could be that the plasmids are copied at different rates. One might be a high-copy plasmid, another might be a medium or even low-copy plasmid.
3. Culture Saturation
Good results are always obtained when preparing the culture. Inoculation from old colonies or over-saturation of the culture will severely negatively affect the plasmid yield. We do not want cultures to be in the late latency phase and we do not want them to be over-saturated.
4. Using Old Colonies
The age of the petri used for starting cultures is important. If your petri is old, even if you have selected a nice big colony, not all of them will be viable cells. Therefore, for best results, purify on a new petri before you start.
5. Antibiotic Problems
Another cause of weak plasmid is antibiotics. Bacteria break down antibiotics as they grow in culture. If not enough antibiotics are added, or if the stocks are old and underpowered, the antibiotic may not be so dominant and you may end up with an antibiotic-free culture.
6. Lysis and Neutralization Problems
Some reagents in lysis are mostly good and stable. But when exposed to air, they can deteriorate over time and may not work as well as on the first day.
One of the biggest mistakes workers make in plasmid isolation is lysis. While protocols normally tell you to be gentle, it's not good to be too precise.
Another common problem is that lysis is allowed to continue for too long, resulting in permanently denatured, indigestible DNA.
7. Isopropanol Quality
Many laboratories have large bottles of isopropanol that are opened and closed throughout the year. If we want to get good yields from the precipitation step, you have to make sure that the isopropanol used is not old.
8. Losing the Pellet
Isopropanol pellets are glassy and clear and difficult to see. A best practice would be to mark the spot where you expect the pellet to form after centrifugation in a fixed angle rotor. This way you know where to find the plasmid when you pour the isopropanol.
Another way to ensure that you know where your pellet is when using a fixed angle rotor is to always load your tubes in the same way, so your pellet will always be in the same place.
!!! Note that many commercial kit manufacturers have developed kits for this problem, using "precipitators" or silica disk filters.
!!! Also a small note: we recommend not to reduce the centrifugation time and speed shorter than optimized. If you cannot increase the centrifugation speed, never shorten the time.
9. Blockage of Columns
If you are worried about clogging the columns, strain through Whatman paper before proceeding to the column step.
10. DNA Remaining in Columns
DNA may remain on the columns after elution. Perform a separate elution to remove any remaining DNA. Using elution buffer heated to around 50°C can also help increase elution efficiency.
References:
Maniatis, T., Fritsch, E. F. and Sambrook, J. Molecular cloning. New York: Cold Spring Harbor Laboratory; 1982. pp 545.
Begbie S. et al. (2005) The Effects of Sub-Inhibitory Levels of Chloramphenicol on pBR322 Plasmid Copy Number in Escherichia coli DH5?
.
Journal of Experimental Microbiology andImmunology (JEMI). 7:82-8.
While DNA can remain stable for many years when stored under appropriate conditions, RNA is short-lived. Because enzymes called "RNase", which can be found in all kinds of conditions, cause RNA degradation. Therefore, a method that can isolate total RNA from cells with high efficiency and at the same time prevent RNA degradation is required.
What is total RNA?
Total RNA is all the RNA molecules present inside a cell. All prokaryotic and eukaryotic cells contain the following types:
Messenger RNA (mRNA): A long protein-coding messenger RNA that carries to DNA and serves as an instant readout of cellular gene expression under certain conditions.
MicroRNA (miRNA): Countless other non-coding RNA molecules, many of which are involved in the regulation and silencing of gene expression.
Ribosomal RNA (rRNA): An important component of ribosomes and critical for protein synthesis.
Transfer RNA (tRNA): Another critical component for protein synthesis. It is formed by processing a precursor molecule in the nucleus. These RNA molecules carry amino acids to the ribosome and base pair with mRNA to ensure that the correct amino acid is added to the synthesized protein.
RNaz What is it?
Ribonucleases (RNases) are enzymes that cleave RNA. These enzymes are very problematic during RNA isolation because they are ubiquitous and very difficult to eliminate. When isolating total RNA you need to make sure that you eliminate the RNAase.
Total RNA Isolation RNases Trizol Removing Using
Inactivating RNases is, of course, not impossible. The first method to safely isolate RNAs while inhibiting RNases was the Guanidinium thiocyanate-phenol-chloroform isolation method applied by PiotrChomczynski and Nicoletta Sacchi. This method is a form of a potent protein denaturing chemical that breaks down protein cell components and inactivates all enzymes including RNases. Isolation typically uses acidic phenol-chloroform to confine total RNA in a clear aqueous phase, while proteins and cell debris end up in the pink organic layer. The RNA can be recovered by precipitation with isopropanol, washed and then redissolved in water. There are many ready-made brands of this method. And it is also possible to make your own phenol guanidine isothiocyanate mixture.
For total RNA isolation Trizol's Benefits
Denaturing RNases.
Isolation of total RNA, including small molecular weight RNAs such as miRNA
High quality RNA.
It is relatively simple to use.
It allows simultaneous isolation of DNA, protein and RNA from a sample.
Trizol Disadvantages of Using
It can be costly compared to other methods of RNA isolation,
Dangerous and toxic,
If not handled carefully, isolated RNA may be contaminated with phenol during phase separation,
It can be time-consuming.
For RNA isolation Trizole Alternatik Commercial Kits Used as
There are many commercial kits that do not use trizol or other hazardous chemicals. Today, commercial kits are available that are suitable for almost all applications. These kits can use spin column or magnetic bead methods to capture RNA. There are also kits specifically designed for miRNA or other small molecular weight RNA species. Kit methods are often safer and more stable than Trizol because they are optimized to extract RNA from different sources and materials.
SUMMARY
Trizol and commercially available kit methods have pros and cons in terms of cost, use of hazardous chemicals and convenience, each of which should be carefully considered before selection.
In operating rooms, doctors can now direct robotic codes with a precision of a few nanometers and operate on patients remotely from computer screens.
Genetic laboratories equipped with DNA splicing enzymes - just a polypeptide chain sequence - can work wonders. The entire genetic makeup of humans can be translated into understandable genetic code.
Medical Biotechnology has advanced by leaps and bounds in the last few years. In this article, we set out to list some of the breakthroughs of biotechnology in medicine.
1. Stem Cell Research
Stem cells can continue to divide indefinitely during the early development of an organism and have the capacity to differentiate into body cells. In a laboratory, researchers can program these stem cells to differentiate into specific cell types. This is where biotechnology innovation comes in. Imagine an individual with a degenerative disc disorder that severely affects their quality of life. With the help of stem cell research, it may be possible to grow these stem cells in vitro in the laboratory and then implant them back into the body of the affected person. This would help them regain their cognitive acuity, vision, hearing and other physical attributes. This sounds a bit like a science fiction movie, but it is promising.
2. Human Genome Project
Often hailed as one of the greatest discoveries in human history, the Human Genome Project (HGP) was an international scientific research project coordinated by the National Institutes of Health and the US Department of Energy. It was launched in 1990 to determine the sequence of nucleotide base pairs that make up human DNA. In April 2003, researchers announced that they had completed the preliminary sequencing of the entire human genome. As researchers learned more about the functions of genes and proteins, it helped them identify the genes that cause diseases.
3. Targeted Cancer Therapies
Standard chemotherapies are toxic to healthy cells. Targeted cancer therapies are drugs that work to minimize damage to healthy cells either by interfering with the function of specific molecules or by targeting only known cancerous cells. According to the National Cancer Institute, "Eventually, treatments could be personalized based on the unique molecular targets produced by a patient's tumor."
4. 3D Visualization for Surgery
Surgery is brutal on the human body and medical breakthroughs that make the healing process more efficient are always welcome. Biotechnology has now made it possible for doctors to view an entire 3D image of the inside of a patient's body using MRI and CT scans. Furthermore, augmented reality will allow relevant information to be displayed directly superimposed on the relevant body parts.
5. HPV vaccine
Human Papilloma Virus (HPV) is one of the causative agents of cervical cancer. It is the second most deadly cancer in women. Therefore, a successful HPV vaccine is considered a major medical achievement. The US Food and Drug Administration (FDA) has approved some PV vaccines for use in women between the ages of 9 and 26.
6. Face Transplant
Face transplantation is the process of using skin grafts to replace all or part of a patient's face with that of a donor. The first partial face transplant was performed in 2005 in Amiens, France. The next successful face transplant was performed five years later in Spain; this was also the first full face transplant. The transplant patient, whose face had been badly damaged in an accident, received a new nose, lips, teeth and cheekbones in a 24-hour operation.
7. CRISPR
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), which we have covered in more detail in previous newsletters, is a relatively new gene editing system that has been hailed as a groundbreaking tool in medical research. By actively testing newly found mutations, researchers can continuously keep pace with genetic mutations to correct targeted therapies
8. 3D Printed Organs
Artificial limbs have been used for centuries and there has been a continuous improvement in the mobility and versatility of bionic limbs. Now new advances in bionic technology and 3D printing have taken it even further. It has made it possible to artificially construct internal organs such as hearts, kidneys and livers. Doctors have been able to successfully implant them in individuals in need.
9. Nerve Regeneration
Nerve damage from neurodegenerative disease and spinal cord injury is known to be largely irreversible. However, researchers have made significant progress in synthesizing rare enzymes that promote the regeneration and growth of injured nerve cells. Neurotrophins are proteins that promote the development of neurons. They are a series of small molecular chains with strong neurotrophic properties. While these neurotrophins have some of the shortcomings of protein-based agents, researchers are pursuing this as a possible pathway for nerve regeneration.
10. Technology to Translate Brain Signals into Speech
Scientists are trying to develop a device that can translate brain signals into voice speech using a voice synthesizer. This would serve as an incredible tool in communicating with individuals paralyzed by disease or traumatic injuries. Scientists have also found that they can use these devices in epileptic patients to isolate the source of their seizures.
Reference: https://explorebiotech.com
Increased thermal stability and hybridization specificity
Proven accurate gene identification in qPCR-dPCR assays
Ability to discriminate between alleles (SNPs) in a single pathway
Easier and more flexible designs for problematic target sequences
LNAs, referred to as 'Locked Nucleic Acids (KNA)' in Turkish, are useful synthetic RNA derivatives. They are also known as bridged nucleic acids (BNA). And it is often referred to as inaccessible RNA. It is bounded by a methylene bridge from 2′-oxygen and 4′-carbon atoms in the sugar-phosphate main line, and this linkage fixes the sugar ring in a 3′-endo conformation that allows the formation of hybrids complementary to DNA and RNA sequences. In this way, LNA is locked in conformation with RNA mimicry. This structure provides increased stability against enzymatic degradation. Furthermore, the structure of LNA has enhanced specificity and affinity as a component of a monomer or oligonucleotide.
LNAs are used in many real-time PCR and digital PCR applications, require no additional equipment and are compatible with all instruments. When incorporated into DNA oligonucleotides, LNA oligonucleotides offer several benefits compared to native DNA bases:
- There is less mismatch discrimination than with conventional probes.
- Incorporation into short DNA primers (< 30 nt) increases Tm by 3-8 °C for each substituted nucleotide. The increased hybridization temperature gives the sequence structural stability.
- It is widely used for specialized applications such as genotyping and transcript variant detection or differential detection of microbial species.
- The high nuclease resistance of LNAs is an important benefit for in vivo and in vitro applications. Numerous studies confirm the superior properties of LNAs as antisense agents
- There are studies with LNA in diagnostic fields.
- Immobilized LNA probes have been successfully introduced in multiplex SNP genotyping assays. This is an indication that LNAs will be much more present on the market in the future.
Every PCR reaction has the same problem:
Very little amplification of target DNA, but more amplification of non-target DNA. However, you can solve the problem of your PCR reactions by using some additional reagents. It is up to you to determine which PCR reagent does what and which condition is best for you.
PCR extension reagents generally work in one of the following two ways:
① By reducing secondary structures and increasing amplification of target DNA ↑
② By reducing non-specific binding and decreasing amplification of off-target DNAs ↓
- Magnesium Effect
Mg⁺² is required for polymerase activity. Withoutsufficient Mg⁺², taq polymeraseremainsinactive.The correctconcentration of Mg⁺² increasesspecificity. LowMg⁺² concentration increasesnon-specific binding.
- BSA
Bovine serum albumin is a commonly used addition in various molecular biology applications, especially in restriction enzyme digests and PCR. In PCR, BSA can help combat contaminants such as phenolic compounds. It is also reported to prevent reaction components from sticking to tube walls.
- Betain
Improves amplification of GC-rich and difficult DNA targets. Ideal for use on sequences known to be difficult to amplify. Reduces DNA Tm temperature.
- Non-ionic Detergents
The use of chemical components such as Triton X-100, Tween 20, NP-40 is also thought to reduce secondary structures. The use of these reagents may increase PCR efficiency but may also increase non-specific amplifications. Therefore, they should be used with caution.
- DMSO
It is thought to reduce secondary DNA structures. Therefore, it may be advisable to add GC-rich constructs during PCR analysis. However, DMSO can also cause a decrease in Taq polymerase activity, so it is very important to balance the two.
- Formamide
It is an organic PCR extension reagent. It works to clean up dirty PCR reactions by reducing non-specific binding.
Although all these components improve PCR results, it is not possible to predict at the first stage what is best for your analysis. It takes a lot of experimentation to optimize a PCR reaction.
Gene drive is a type of genetic engineering technique that alters genes so that they do not follow typical rules of inheritance. It overrides natural selection by replacing a natural gene with a new gene that is then passed down from generation to generation.
Due to the potential uses and implications of gene drive, there is a growing interest in this technology internationally.
How does it work?
The gene drive consists of three key components:
- the gene you want to propagate,
- the Cas9 enzyme that can cut DNA,
- CRISPR, which determines where the enzyme should cut.
The genetic material encoding these three elements is inserted into an animal's DNA to replace the natural gene you want to replace on both chromosomes. Thanks to CRISPR-Cas9, a gene editing technology that utilizes bacteria, it is becoming easier for researchers to create gene drives.
NOTE
To update your information about CRISPR-Cas9 Letgen BiotechnologyPublished by Mart You can review the bulletin of the month.
When an animal carrying the gene drive mates with an animal not carrying it, it receives a copy of DNA, a "natural version" and a "gene drive version". When the chromosomes line up for the first time after fertilization, CRISPR in the gene driver DNA is activated and the DNA-cutting enzyme Cas9 is directed to cut the copy of the natural version before development begins. When the natural gene is cut, the cell's repair mechanisms are triggered and the damaged DNA is restored. But it uses the chromosome carrying the gene drive as a template. In the end, when the repair is finished, both chromosomes carry a copy of the gene drive.
This is how it is passed on from generation to generation. And so the process continues...
Two scientists, Crisanti and Burt, had been studying mosquitoes for years. They wanted to bypass natural selection and introduce a gene that spreads very quickly through mutations. Their goal was to destroy mosquitoes by preventing them from spreading disease. In the end, a gene they inserted into the mosquito genome traveled through the population, reaching more than 85% of the generation.
But scientists are still working to determine the ecological and environmental impacts of using gene drives to eliminate an entire species.
Is it dangerous?
Several surveys in the US have shown that the majority of the public would support the use of agricultural gene drives against pests if provided with sufficient information about the risks and benefits of the technology.
It is still difficult for scientists to isolate the ecological impacts of gene drive projects.
Work is also ongoing on potential remediation plans or remediation strategies to remove the gene drive from the environment in the event of unintended consequences.
While gene drives hold promise for ensuring human health and ecological balance, there is still a long way to go on the implications and effectiveness of the technology.
PCR success can vary depending on your experimental design.
Small errors can often result in low yields or false negative/positive products.
4 essential tips to ensure the success of your PCR reaction:
1. Primer Design
- Find the optimal primer concentration.
- To avoid secondary structure formation, a primer 18-30 nucleotides in length and a GC content of 40-60% is recommended.
- Check primer homology. Any binding between your primers or internally will result in reduced PCR efficiency.
- The binding degrees of the primers are calculated with the formula 2 (A+T)+4 (G+C).
- Finish with a G or C. Closing the 3′ end of your primary knee with a G or C will strengthen the primary extension at the extension site.
2. Target Sequence
- A PCR reaction can be affected by both the quality and quantity of your DNA template.
- A quality DNA sequence will increase the specificity of the reaction and product yield.
- Be careful to avoid contamination! Protein or chemical contamination can cause non-specific binding or completely inhibit PCR.
- Check that the 260nm/280nm ratio of your DNA absorbance is ≥1.8.
3. Reaction Reagents
- Hazardous reagents affect the efficiency of your PCR reaction. Although many laboratories use commercial off-the-shelf components, the components to know and consider are:
DNA Polymerase, dNTP, magnesium concentration.
4. Thermal Profile
- Thermocycler protocols and conditions are highly standardized for PCR. Here are three main things to consider:
" Modified PCR conditions:
" Bonding Temperature: it would be optimal to set it 3 °C below the melting temperature of your primary sequence. For further optimization the anneling temperature can be increased in steps of 1-2 °C.
" Extension time: It is recommended to set an extension time of 1 minute for every 1 kb amplicon. Optimal extension rates may depend on the processability of the DNA polymerase.
Primer/Probe What is it?
Primers (Oligonucleotides) are short sequences of nucleotides. They are used in many molecular and genetic procedures. Nucleic acid probes are very important reagents for detecting specific nucleic acid sequences by hybridization. Visualization of a specific hybridized probe in solution or added to the solid phase is the basic principle of the hybridization assay.
The specificity of the primers and probes to the oligonucleotide sequence should be checked by quality control tests. Standard desalted (DSLT) purification and HPCL purification options can be offered as primer purification processes. Whichever purification method is preferred, the consumption will vary according to the final concentration used in the PCR process. Therefore, the estimated number of PCR reactions given according to the synthesis scale prepared varies considerably.
Lyophilized primer/probelar can be stored at room temperature as it is not affected by temperature changes. After reconstitution, it should be stored at -20 ºC. For long-term use, it should not be freeze-thawed and should be aliquoted.
Primer/Probe How to Reconstitute?
TE (Tris-EDTA buffer solution) or DNase/RNase free water (DNase/RNase free water/DEPC water) is used to solubilize oligonucleotides.
- 100 μM = 100 μMoles/L = 0.1 nMoles/μL
- Master stock = 100 μM
Amount of Oligo
34.0 = 155.5 = 0.96
OD 260 nMoles mg
- 100 μM = X nmol lyophilized primer + (X × 10 μl H2O)
- For example , if 155.5 nmol of primer is present, 1555 μl H2O is added to create a 100 μM primer master stock.
- If working stock = 10 μM
- Dilute the primer master stock with H2O 1:10 in a sterile microcentrifuge tube.
Transcription Activator-Like Effector Nucleases
TALEN was first discovered in 2009 and successfully used for genome editing work in 2011.
Proteins called TALENs (Transcription Activator-like Effector Nucleases) function as transcription factors that turn on genes in plant hosts that support their successful infection.
TALENs are natural proteins made and used by harmful plant bacteria to control plant genes during infection.
TALEs represent a family of type III effector proteins from Xanthomonas spp., a genus of gram-negative Proteobacteria often associated with plant diseases. These intelligent microbial creatures have developed their own way to control gene expression in plants, primarily through TALEs.
Figure: Schematics for the reconstitution of TALE. The central binding domain contains multiple regions of repeated amino acid sequences (positions 12 and 13 are the most important for specific nucleotide recognition). DBD, DNA binding domain. FokI, Fok 1 nuclease.
Structure
A TALE is made up of successively repeated segments, each usually consisting of 34 amino acids. Each of these pairs of amino acids binds to a specific nucleotide in the DNA. This usually enhances the transcription of that gene.
If the nucleotide sequence of the region to be modified is known, a TALE can be synthesized by combining the corresponding repeat sequence. To this construct, an endonuclease is added that can cut the DNA to which the TALE binds.
Once TALEN enters the nucleus of the cell whose genome you want to change, it cuts the DNA creating a double-stranded break (DSB). This can be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR).
The key technology behind TALEN is the use of transcription activator-like effectors (TALE) to bind a specific DNA sequence. When combined with a nuclease such as FokI, the precision of the cutting site becomes extremely efficient.
Applications
- So far, TALENs have been used to alter genes in many species: plants, animals, fungi and monkeys.
- In vitro mutagenesis studies using cell lines
- In vivo genome editing
- Sugarcane for use as biofuel
- Crop improvement applications
TALENs How to use it to improve our crops?
TALENs are used by scientists to help improve food crops.
TALENs are a tool used to extend the characteristics of crops and allow breeders to selectively modify the qualities they want; such as increased yield and quality, tasty foods, resistance to disease. Resistance to environmental factors.
To date, TALENs have been able to produce soybeans that produce superior quality oil, rice that is more aromatic and resistant to bacterial blight, potatoes with better taste and less carcinogenic acrylamide, and wheat that is fully resistant to powdery mildew.
With the need to double food production to feed a projected population of 10 billion by 2050, TALENs and other new breeding innovations are essential tools to ensure food security.
Outside of plants and algae, TALENs have been successfully used to modify genes in yeast, fruit flies, roundworms, crickets, zebrafish, frogs, rats, pigs, cows, silkworms and humans.
Conclusion
TALENs remain the most specific gene editing tool today.
TALENs are highly precise gene editing tools that can target any sequence and in some cases outperform CRISPR.
However, the technology requires the creation of engineered proteins.
While the technology is well established, proven and specific, protein engineering is time consuming and expensive. Only a fraction have high performance. And simultaneous editing is difficult.
TALENs are great for molecular genome engineers and newer and better ways of using TALENs will emerge every day.
What is CRISPR-Cas9?
Although the existence of CRISPR clusters has been known since the 1980s, their role in the defense mechanism of living beings has been proven relatively recently. Since 2013, the technology of gene editing using the CRISPR-Cas9 system, which has been the center of attention of the entire scientific world, has been described by Science magazine as one of the fastest, most successful, cheapest and highly accurate methods of genome modification that humanity has discovered to date.
CRISPR (clustered regularly interspaced short palindromic repeats = clusters of regularly interspaced short palindromic repeats) /Cas9 (CRISPR-associated nuclease-9) are prokaryotic DNA segments containing short repeated base sequences. All archaea and half of bacteria have this system.
How does CRISPR-Cas9 work?
There are two important molecules that create the mutation:
1. An enzyme called Cas9: It acts as a "molecular scissors" that can cut two strands of DNA at specific places in the genome. This allows DNA fragments to be added or removed.
2. Guide RNA (gRNA): Consists of a small (about 20 bases) pre-designed RNA sequence inside a longer RNA skeleton. The long RNA skeleton binds to the DNA and the predesigned sequence guides Cas9 to the right spot in the genome. The Cas9 enzyme then cuts in the right places.
- A Protospacer Adjacent Motif (PAM) must be present in the target gRNA.
- It enables the binding of the gRNA complex to DNA, which forms a ribonucleoprotein complex with the Cas9 enzyme via the PAM sequence.
- Cas9 follows the guide RNA to the appropriate location in the DNA and makes a cut in both strands of the DNA.
- At this stage, the cell realizes that the DNA has been damaged and tries to repair it.
- Thus, using the DNA repair mechanism, it can modify one or more genes of the cell of interest.
What does the future hold?
- It has high potential in the treatment of many diseases such as cancer, HIV, Parkinson's, malaria, hepatitis, vision loss correction, which are very difficult to treat and whose mechanisms are unknown. To date, this system has been utilized in the treatment of diseases, albeit to a lesser extent.
- It is known to be much easier, faster and less costly than other gene editing systems.
- Since the possibility that changes to reproductive cells could be passed on from generation to generation would trigger ethical issues, it seems a long time before it can be used on humans.
- There are many efforts to eliminate "off-target" effects. Even though the system is specific, there is still the possibility that in some cases it may make a mistake by cutting at a different point instead of the targeted gene.
- The 2020 Nobel Prize in Chemistry has been awarded many prizes, including the Nobel Prize in Chemistry, but the success rate of the system has still not exceeded 30%.
Conclusion
CRISPR/Cas technology is becoming increasingly precise and efficient and will continue to be used as an effective tool for functional genomics studies and for improving the properties of products.